Proteins that terminate with a "CAAX" sequence motif (e.g., the Ras proteins, Rho proteins, and nuclear lamins) undergo three sequential posttranslational modifications. First, the cysteine (C) is isoprenylated by enzymes in the cytosol (farnesyltransferase or geranylgeranyltransferase I). Second, the last three amino acids of the protein (i.e., the -AAX) are released by Rce1, an integral membrane endoprotease of the endoplasmic reticulum. Third, the newly exposed isoprenylcysteine is methylated by isoprenylcysteine carboxyl methyltransferase (Icmt), also an integral membrane protein of the endoplasmic reticulum. Each of these modifications is important for the targeting of CAAX proteins to membrane surfaces and for their proper function. For example, inhibitors of farnesyltransferase block the membrane targeting and downstream effects of mutationally activated Ras proteins and have shown promise in the treatment of certain cancers. A potential therapeutic drawback of the farnesyltransferase inhibitors is that K-Ras, the Ras isoform most commonly involved in human cancers, is geranylgeranylated by geranylgeranyltransferase I in the setting of farnesyltransferase inhibitors. The existence of this alternate prenylation pathway has raised interest in defining the physiologic effects of blocking the enzymes involved in the "post-isoprenylation" processing of CAAX proteins (Rce 1 and Icmt), as those enzymes act on both farnesylated and geranylgeranylated proteins. The goal of this project is to gain insights into the potential therapeutic utility of interfering with protein processing by Icmt and Reel, using cell culture systems and genetically modified mice. The effects of a deficiency in Fntb (the gene encoding the beta-subunit of protein farnesyltransferase) will also be assessed. Many useful reagents have already been generated for this project, including mice with knockout and conditional alleles of lcmt, Reel, and Ftnb. Preliminary studies have uncovered many exciting findings, including the fact that Icmt deficiency strikingly reduces oncogenic transformation of fibroblasts by K-Ras. Specific Aim 1 of this project is to assess the physiologic impact of Icmt deficiency, further defining its effects on Ras transforming activity. Specific Aim 2 is to generate cell culture and animal models for Fntb deficiency, and to compare the impacts of Fntb deficiency, Icmt deficiency, and Rcel deficiency. Specific Aim 3 is to determine if deficiencies in Icmt and Reel render cells more sensitive to farnesyltransferase inhibition. These studies should define the therapeutic potential of blocking the three different steps of CAAX protein processing.